PROJECT SUMMARY-ABSTRACT Our goal is to generate and validate an innovative 3D cellular model of the nervous system from human pluripotent stem cells as a new tool for aging research. The current state of the art for in vitro modeling is insufficient because multicellular human neural circuits in culture conditions remain in immature states for prolonged periods of time. To overcome this barrier, we are proposing to produce a Microassembly of Bioengineered, Rapid, All-Inducible Neural System (BRAINS). We will utilize our recently optimized protocols to produce consistent 3D cocultures generated from novel transgenic cell lines with transcription activator-like effector nuclease technology that we have generated in preliminary data. In Aim 1 neurons, astrocytes and oligodendrocytes will be individually produced from human pluripotent stem cells by induction of neurogenin 2, Sox9 in combination with NFIA, and Sox10, respectively. We will assemble these cell types together in defined 3D coculture spheres and assess neural circuit maturation over time. Maturation will be determined by increased presence of myelinated fibers and onset of synchronous network activity. Activity will be measured by multielectrode array recordings as well as live calcium indicator imaging. These studies will determine the optimal conditions for producing the functional BRAINS. In Aim 2, we will test whether regulated neuronal activity will enforce maturation in a rapid and consistent manner. We will utilize a novel optogenetic cell line generated in preliminary studies to determine the effect of light-mediated stimulation of neurons upon circuit maturation within the BRAINS. The results from these studies will reveal if evoked neuronal activity accelerates production of mature circuits. In Aim 3, we will test whether forced expression of the transcription factor NFIX in astrocytes will induce aging phenotypes by performing assays for cellular aging and by measuring the effect upon myelination and neural network activity of cocultured cells. Altogether, we expect to deliver novel cell lines and an optimized protocol to systematically generate a human neural circuit model system with aging phenotypes. The BRAINS can be a breakthrough tool for use throughout the research community for disease modeling and screening drug interventions in the context of age-related conditions such as Alzheimer?s disease.